Ion Mobility Spectrometry (IMS) followed by Mass Spectrometry (MS) analysis is an emerging and very powerful technique that provides extra structural information, and an increased resolving power, both these features being very useful in the fields of -omic, which include metabolomics studies, proteomic studies and other biological analysis, and petroleomic studies, as shown by different studies1-5. There are, to date, different IMS approaches:
1) Drift Time IMS (DT-IMS)6 is one of the best known mobility techniques, perhaps due to its simplicity, robustness, speed, and relatively small size and power consumption. DT-IMS are mostly used for military and security purposes7, although they are also used in other industries as well as in many new areas of research including proteomics and structural biology8-11. The resolving power of DTIMS (R) is mainly limited by Brownian diffusion; classic DT-IMS can reach R=100, but their sensitivity is limited by a low duty cycle. Nevertheless, their transmission can be improved by the use of ion funnels12, multiplexing and ion accumulation13. Resolving powers higher than 300, and approaching 400, were achieved with the so-called High-Resolution Ion Cyclotron Mobilityl14,15. The pulsed input and output of DT-IMS might be advantageous if the ion source is also pulsed, but it usually hinders transmission and complicates the interfaces in tandem schemes, such as IMS-MS, and with other continuous ion sources such as electro-spray (ESI).
2) Travelling Wave IMS (TW-IMS): Its separation mechanism allows for true mobility separation, but in practice it also produces pulsed packets of ions and, what is more serious, the reliability of the structural information obtained is unclear because: (i) in the intense electrical fields required, ion heating can have a significant effect16, and (ii) drift time is related to the mobility in a complicated way, which is still not completely understood17,18.
3) Field Asymmetric IMS (FAIMS)19-22, also termed Differential Mobility Spectrometry (DMS), is an alternative and robust technique that separates ions in space rather than in time, thus producing a continuous flow of selected ions with a 100% duty cycle. FAIMS separates ions according to nonlinearities in the mobility arising in strong fields23-25, and traditionally produced relatively poor resolving powers (near 20). Nevertheless, recent developments26-29 have shown that the separation capability can be dramatically increased by adding polar dopants to the drifting gas. Shvartsburg and Smith30 also reached resolving powers exceeding 200 by increasing the time of residence of ions within the filter. The new generation of DMS-MS commercialized as ‘SelexIon’ is a powerful tool to reduce background levels31, and allows mobility selection before ions pass through the Atmospheric Pressure Ionization (API) interface32, which permits the incorporation of the IMS by a relatively simple upgrade of the MS (if compared with TW-IMS that require low pressures), but it does not provide clearly interpretable structural information.
4) Differential Mobility Analysis (DMA) provides absolute mobility analysis, and also produces a continuous output of mobility-selected ions. Planar DMAs33 permit coupling with virtually any API-MS33 and provide an improved transmission of ions. The mobility is measured at moderated ionic temperatures with little fragmentation, which makes structural interpretation of the data easier34-37. However, DMAs require a flow with high speed and high Reynolds numbers (Re) that is prone to turbulence38.
5) Variable Electric Field Mobility Analysis (VEFMA) US 20100243883 A1 also provides a continuous output of mobility selected ions. Ions are separated according to their true mobility using only electric fields. The selected ions coalesce at the analyzer outlet while other ions are deflected away and not transferred. Ions are separated in space and thus a continuous flow of filtered ions with a narrow range of selected mobility ions is produced, as in Differential Mobility Analyzers (DMAs); yet no high fluid velocity field is required, thus avoiding the limitations in DMAs associated with flow unsteadiness, and turbulent transition. VEFMA is at present the only technology capable of: (i) producing a continuous output of mobility selected ions, (ii) operating at ambient pressure, (these two aspects are essential for the Add-on architectural capability), (iii) selecting the ions according to their absolute mobility, and (iv) being able to operate in transparent mode (i.e. allowing ions of all mobilities to pass though the outlet of the analyzer without being mobility selected).
Tandem IMS-IMS: While IMS is very powerful, tandem IMS-IMS analysis and pre-filtration is also attracting increasing interest. In the general IMS-IMS scheme, two mobility filters are coupled in series, and ions are preselected according to their mobility in the two different stages. As illustrated by the pioneering work by Clemmer's group39-43, IMS-IMS-MS analysis provides an extra dimension of separation, which increases the total separation capacity42. The recent study by Hill's group44, where a Drift Tube IMS (DTIMS) was coupled with a Synapt MS (from the commercial brand Waters), also illustrates the potential of the (IMS-MS)2 approach. This set-up, for which the outlet of the DTIMS was also gated to pass only one type of mobility selected ions, also shows that, if a high duty cycle is required, it is very desirable to use mobility pre-filter capable of producing a continuous output of mobility selected ions when coupling with pre-existing mass spectrometers.
The use of pulsated output IMS analysis techniques (namely DTIMS and TWIMS) for IMS-IMS analysis has two main problems:                i) the duty cycle of each stage is usually very low (around 1%), and the duty cycle of the composed architecture tends to be even lower (1% times 1%=10−4), and this low duty cycle reduces the sensitivity of the analyzers.        ii) Coupling the two pulsed IMS stages requires a complicated synchronization to gate the desired pulse of ions in the second stage at the time when they arrive at the outlet of the first stage, which is not known a priori.        
For these reasons, it is more desirable to use mobility filters that produce a continuous output of selected ions, such as FAIMS, DMA, and VEFMA. Although these technologies are historically grouped together, a main aspect differentiates FAIMS from DMA and VEFMA: FAIMS measures the variation rate of the ion mobility at increasing electric fields (it does not measure the mobility of the ions), while DMA and VEFMA measure the absolute mobility of the ions (defined as the ratio of electric velocity to electric field).
Tandem DMA-DMA: The use of tandem DMA systems is described by F. de la Mora et. al. in U.S. Pat. No. 7,855,360 and in its continuation patent U.S. Pat. No. 8,278,622. In this invention, F. de la Mora describes how to operate at least one DMA in tandem with other ion filters, and he highlights the advantage of using various filters in series, including at least a DMA, in which the DMA provides a continuous output of mobility selected ions. In the type of configurations described by F. de la Mora, the sensitivity for targeted ions can be much better than that achievable by traditional IMS in tandem with mass spectrometry approaches (namely, DT-IMS in tandem with MS) because each ion can be monitored with a very high duty cycle. F. de la Mora proposes various approaches to the pre-filtration of ions in two DMA in tandem, which are relevant to the present invention: (i) By operating each DMA at different speeds, the invention of U.S. Pat. Nos. 7,855,360 and 8,278,622 allows the mobility to be measured at different electric field strengths, which, as described by F. de la Mora, allows the separation capacity of the tandem DMA-DMA architecture to be increased. Alternatively, the invention of de la Mora also incorporates using two DMA in tandem, and additional means to change (by attachment of vapor molecules, or by fragmentation, or by oxidation) the ions after being analyzed in the first DMA, and before entering in the second DMA. This approach was previously used by McMurry and colleagues at U. Minnesota45 for the analysis of aerosols, but F. de la Mora extended the concept to the analysis of ions.
The architecture described in U.S. Pat. Nos. 7,855,360 and 8,278,622 allows for the detection of one or several target ions within a mixture of ions with high resolution. However, when the filtering parameters of each DMA are not known a priori, which is a very common circumstance if the operator wishes to identify these variables, or if the operator wishes to re-calibrate the instrument, all the DMA stages must be scanned together in a multi-dimensional spectrum because they cannot operate in transparent mode, and this scan can be very time consuming. The typical identification of the filtration parameters in a triple quadrupole comprises the following steps: (i) in order to calibrate the first quadrupole, a known amount of substance is introduced into the instrument by the operator, the third quadrupole is operated in transparent mode (allowing the passage of all ions), the first quadrupole is scanned so as to produce a spectrum, and the mass of the precursor ion is identified in this first spectrum. Once the precursor ion is identified, in a second step (ii), the first quadrupole is operated to pass only the selected precursor ions, and the second quadrupole is scanned so as to produce a spectrum, which is used to identify the masses of the product ions. This procedure is relatively quick because the two spectra are one-dimensional (meaning that only one parameter is scanned at a time). If each spectrum is composed of 1000 positions of the corresponding filtration parameter, and the measurement of each position takes 50 ms, the time required to identify the filtering parameters in each quadrupole would then be 100 seconds, and the total time for two of these filters capable of operating in transparent mode would be less than 2 minutes. The equivalent procedure in the case of including a DMA and a triple quadrupole takes much longer because the DMA cannot be operated in transparent mode, the identification of the precursor ion requires a two-dimensional scan of the DMA and the first quadrupole, in which the third quadrupole can be operated in transparent mode. If each DMA scan requires 200 points (which are required to have at least four points per peak-width for a resolving power of 50), and the quadrupole scan requires 1000 points, each taking 50 ms, the time required to perform the required double scan is 200×1000×50 ms=10000 seconds (approximately two and a half hours). In an architecture comprising only two DMAs in tandem and a detector requiring 50 ms to measure the signal produced at each point of the spectrum, the time required to identify the position of the peak would be 200×200×50 ms=2000 seconds (slightly more than half hour). The time required to identify the position of the peaks in a DMA-DMA-quadrupole architecture would be simply prohibitive (approximately three weeks).
These times are not a big problem if the system is used to detect species which are previously known. For instance, the architecture can be used in an explosive detector, for which the filtering parameters are not expected to change (aside from fine tuning), but these high times become a real problem if the architecture is to be used in a more general purpose platform for which identification of the filtrating parameters can be a regular procedure. Accordingly, one objective of the present invention is to teach how to operate a set of ion filters, in which at least one of them is and ion mobility filter, and in which at least one of said ion filters can be operated in transparent mode (allowing the passage of all ions) so as to reduce the time required to identify the peaks of the species of interest.
Having a good transmission is important if the user wishes to detect species for which their properties (and hence the position of the peaks in the spectra) are previously known. For instance, if the two DMA in tandem are to be coupled with a mass spectrometer as described in U.S. Pat. Nos. 7,855,360 and 8,278,622. On the other hand, the possibility to operate in transparent mode (allowing all ions to be transferred together irrespectively of their mobility), which is offered by the VEFMA, provides a higher flexibility and shorter peak identification times, which greatly facilitates the identification of the peaks in the stages of analysis by reducing the time required to complete the identification (from 2 hours to 3 minutes).
The invention described in U.S. Pat. Nos. 7,855,360 and in 8,278,622 also has the problem that transmission of ions between one DMA and the next is very poor. Each DMA requires a laminar and high speed flow (with high Reynolds and high pressure gradients) to separate the ions, and these flows are very delicate because they easily become turbulent due to their high Reynolds. This problem can be solved by means of using very carefully designed DMA drift flow channels, which remain laminar at very high Reynolds by maintaining the boundary layer of the DMA flow constantly accelerated and unperturbed. However, the strong pressure gradients produced by the high speed flow tend to deform the inner walls of the DMA, and these deformations affect the boundary layer of the flow, which might easily become turbulent, thus destroying the resolving power of the DMA. A solution to this problem is explained by Rus et al. (See US 20080251714), where a rigid structure is used to minimize deformations and to ensure that the whole structure is gas tight, such that the boundary layer in a DMA remains unperturbed. Rus also teaches how to transfer the ions from the DMA to a mass spectrometer, where the ions are directed towards the MS by the gas that passes from the DMA toward the vacuum side of the MS at very high speeds, as they are suctioned by the vacuum of the MS. However, if the rigid structure of US 20080251714 is used in combination with the tandem DMA-DMA architectures proposed in U.S. Pat. Nos. 7,855,360 and in 8,278,622, then the transmission of ions would be very poor for two main reasons:                (i) in a tandem DMA-DMA scheme, the local pressure gradient that pushes the gas and the ions from one DMA to the next cannot be very high, because the flow of incoming gas and ions that pass from one DMA to the next would otherwise form a jet in the second DMA that would perturb the high Reynolds flow of the second DMA, which would become turbulent, and which would thus have a very poor resolving power and a poor transmission. As a results, ions have to be transnsported from one DMA to the next at low velocities, for which diffusional losses dominate.        (ii) the need for thick and rigid structures in each DMA inevitably requires a thick wall between each DMA, which must be crossed by the slit that allows for the passage of ions from one DMA to the next, resulting in a long time of residence of the ions through these slits. These long slits (the slits are long along the direction of the movement of the ions through the slit) also impede the passage of electric fields, which cannot be used to push the ions forward.        
As a result, the ion losses in the channel that communicates one DMA with the next are very high. Moreover, if additional means to change (by attachment of vapor molecules, or by fragmentation, or by oxidation) the ions after being analyzed in the first DMA, and before entering in the second DMA are used, the ion losses through said long slits (the slits are long along the direction of the movement of the ions through the slit), and through said additional means become even higher.
Tandem IMS-IMS by Means of a Multi-Stage VEFMA: FIG. 1 illustrates schematically one embodiment of a Two-stages 2D-VEFMA, as described in US 20100243883 A1. This embodiment of the VEFMA is composed of two insulator boxes, the first insulator (1) housing the inlet electrode (2), each insulator box housing two deflector electrodes (3), and the second insulator (4) housing the outlet electrode (5). The intermediate electrode (6) is a thin plate that separates the two stages and allows ions to be transferred through the intermediate slit (7). In contrast with the DMA-DMA architecture, the pressure gradients in the VEFMA are very low, and this allows the intermediate electrode (6) to be very thin. As a result, the ions can be transmitted through the slit (7) by the local electric fields that easily pass through the slit. The outlet electrode incorporates a slit (8) which is elongated on the side receiving the selected ions, and which, if required, becomes a rounded orifice on the opposite side of the outlet electrode so as to better fit the inlet of a subsequent analyzer (which can be a mass spectrometer). Ions reaching the outlet slit are directly carried by the flow toward the subsequent analyzer (9), while a counterflow gas (10) exits through the inlet slit (11) so as to prevent droplets from entering the analyzer. The required gases are introduced into each VEFMA chamber through two lateral inlets (12). The voltage required by the inlet electrode (2) is AV1, the voltage of the intermediate electrode (6) is AE2, and the voltage of the outlet electrode (5) is AV3. The Deflector Electrode (3) voltages are DV1 through DV4 (DV1 and DV2 in the first stage (1), and DV3 and DV4 in the second stage (4)).
The usefulness of the analysis of the mobility of the ions in two consecutive stages, which in general is measured by the separation capacity of the system, depends on the statistical orthogonality (or dispersion) of the two measurements. If the mobility of the ions in one stage is linked with the mobility in the second gas, then adding the second stage will not increase the separation capacity, whereas the species will be more separated if the mobilities are more orthogonal, and hence the separation capacity of the system will be higher. Note that separation capacity is defined in the context of the present invention as the number of different species that can be differentiated in a spectrum. The separation capacity will thus be higher if the width of the peaks produced by the analyzer is smaller, and it will be also higher if the dispersion of the physical parameters being measured is higher. FIG. 2 illustrates schematically two sets of mobility pairs, in which each point corresponds with the pair of mobilities of a given ion in each mobility measurement stage. In the first case (left, poor orthogonality), the mobilities in the two stages are linked (the dots are not dispersed, meaning that the measurements are not orthogonal). As a result, two different type of ions, which have the same mobility in the first stage, and which thus pass together through the first stage at a given mobility (not being differentiated in the first stage), cannot be differentiated in the second stage because their mobilities in the second stage are also very similar, meaning that the separation capacity of the analyzer is poor. In the second case (right, better orthogonality), the mobilities in the two stages are not linked (the dots are dispersed, meaning that they are highly orthogonal). In this case, although two type of ions, which have the same mobility in the first stage cannot be differentiated in the first stage, they will be differentiated in the second stage. As a result, the separation capacity of the analyzer is much improved. Note here that FIG. 2 is here used to illustrate how the simultaneous measurements of ions in two different mixtures of gases and dopants can improve the separation capacity, even if the peak width is not affected.
In short, in order to achieve a high separation capacity, it is very desirable to have highly orthogonal measurements. This can be done by modifying the ions between one mobility measurement stage and the subsequent mobility measurement in an intermediate modification cell, which resembles the role of the collision cell in triple quadrupoles. For instance, this type of modification can be achieved by means of an ion funnel, which can be located between the two subsequent drift cells, and in which ions are excited prior to entering in the next stage, as described by Clemmer39,46. Other approaches, as described in U.S. Pat. Nos. 7,855,360 and 8,278,622, would incorporate means between one stage and the subsequent stage such that ions and charged particles undergo some change after being classified in the first filter and before entering in the second filter.
These means can include attachment of vapor molecules, fragmentation, and oxidation. While these approaches increase the separation capacity, they have a poor transmission of selected ions. This poor transmission is mainly caused due to losses of ions in the required ion modification stage. In order to maximize the transmission, it is desirable to pass the ions directly from the one stage to the next, but this scheme would not provide enough space for the required modification cell. In short, the present state of the art imposes a trade-off between separation capacity/orthogonality, and ion transmission. Accordingly, one objective of the present invention is to provide highly orthogonal mobility measurements with a high transmission of the selected ions.
An attempt to solve this problem is described in US 20100243883 A1, in which it is described that each of the two stages of the 2D-VEFMA can be operated with different gasses, such as N2, which is cheap to produce, or CO2 or SF6, such that the first stage provides the measurement of the mobility in one gas, and the second stage provides a measurement of the mobility in a different gas. This architecture offers a good transmission (the duty cycle is 100%, and the ions can pass from one VEFMA stage to the next stage very quickly). However, experimental data shows that the mobility in the different gases (N2, CO2, SF6) is poorly orthogonal. The orthogonality of the measurements in US 20100243883 A1 is poor, and hence the separation capacity of the tandem IMS-IMS analysis is also poor. Besides, the invention of US 20100243883 A1 does not disclose how to identify the position of the peak in each stage independently. Accordingly, one objective of the present invention is to teach how to identify the filtering parameters of a sequence of ion filters, in which at least one ion filter is an IMS, and in which at least one ion mobility filter is of the type that produces a continuous output of mobility selected ions, and that has the capacity to operate in transparent mode (such as the VEFMA). Also, the invention of US 20100243883 does not teach how to control the concentration of different gasses in each stage, which would vary according to US 20100243883 because each stage is communicated with the next though the intermediate slit (7). This slit is required to allow the ions to pass from one stage to the next, but it also allows the gasses to pass from one stage to another. As a result, the composition of the gasses in each stage is an uncontrolled mixture of different gasses initially introduced each stage, thus leading to non repeatable and difficult to interpret results. Accordingly, one goal of the present invention is to enable the passage of ions from one stage to the next, and to control at the same time the concentration of gasses in each stage.
The Use of Dopants in IMS: The addition of polar and non-polar dopants to the gas through which the mobility of ions is measured serves to modify the mobility of the ions, to enhance the signal produced by some desired ions, and to eliminate the signal produced by some undesired ions. Although the mechanisms by which dopants affect the mobilities of the different type of ions is not well understood, the use of dopants is very common in Drift Tube IMS, as illustrated by multiple patents in the field: U.S. Pat. No. 8,237,110B2, US20120138783A1; U.S. Pat. No. 8,084,000B2; US20110300638A1; US20110297821A1; US20110291000A1; U.S. Pat. Nos. 7,999,224B2; 7,994,475B2; 7,985,949B2; 7,956,323B2; US20110114210A1; US20100308216A1; US20090179145A1; US20090039243A1; US20090032699A1; U.S. Pat. Nos. 5,283,199A; 5,234,838A; 5,095,206A. Note that, in these applications, dopants do not increase the overall separation capacity of the instruments. The dopants have the capacity to shift the position of the peaks in mobility spectra. While these shifts can be helpful to improve the sensitivity for some specific species, or to separate some species from their contaminants in some specific scenarios, dopants can have the opposite effect on other species. For instance, due to its capacity to modify the mobility of the ions, a dopant can be useful to separate two species which would otherwise appear at the same mobility if the dopant was not used. However, other analytes, which are properly separated without the dopants, could appear at the same mobility due to the addition of the dopant. In this second case, the introduction of the dopant would be counterproductive. As a result of this, the selection of the right dopant is very application-specific. And hence, the use of dopants, and the selection of the right dopant require specific studies for each application, and cannot be used by default in a general purpose system. An architecture for which dopants could statistically provide an improved separation capacity regardless of the particular application, and for the majority of species of interest (say more than 10% or 20% or 50% or 70% of the species in a sample) would allow users to incorporate the use of dopants in a general purpose system, and would allow them to minimize the required specific studies. However, there is to our knowledge not a solution to solve this problem. Accordingly, one objective of the present invention is to use dopants to statistically increase the separation capacity (regardless of the particular application) in a sequence of filters, incorporating at least two ion mobility filters, and.
It is noted here that dopants are used to enhance the separation capacity in FAIMS (also termed DMS) (US20100308216A1), where they show that the nonlinear effects on the mobility are highly increased. However, FAIMS do not provide absolute mobility ion selection.
Despite the potential offered by dopants, using them in IMS-IMS applications is very complicated. Very small concentrations of dopants (in the ppm range) can produce a very dramatic change in the mobility of certain ions. While this can be a great advantage to enhance the separation capacity of tandem IMS-IMS schemes, including the combinations of VEFMA and DMA, introducing different dopants in each IMS stage has two main problems:    (1) The gases of the different stages tend to pass from one stage to the other because pressure gradients among the different stages tend to drive gases through the ports intended originally to allow for the passage of the selected ions.    (2) Even if one could eliminate these pressure gradients, trace amounts of the dopants would tend to diffuse and pass from one stage to the other.
As a result of these effects, the concentration of dopants becomes unpredictable and difficult to control. And hence the mobility varies in an uncontrolled fashion, which makes it impossible to take advantage of the use of dopants in IMS-IMS schemes. Accordingly, one objective of the present invention is to control the concentration of dopants in a sequence of ion filters incorporating at least one ion mobility filter.
In conclusion, for the analysis of the mobility in various IMS analyzers operating in series, it is very desirable to be able to:    (i) provide a high resolving power,    (ii) provide a high ion transmission,    (iii) measure the absolute mobility    (iv) provide the possibility to operate each IMS stage in transparent mode, and    (v) utilize mobility measurements in each stage that provide a high orthogonality among stages, and which thus provide an improved separation capacity.
Accordingly, one goal of the present invention is to solve the problem of producing highly orthogonal IMS-IMS measurements with various mobility filters in tandem.
Another objective of the present invention is to accomplish highly orthogonal mobility measurements and a high transmission of the selected ions.
A further objective of the present invention is to increase the separation capacity (regardless of the particular application) in a sequence of filters, incorporating at least two ion mobility filters.
Yet a further objective of the present invention is to accurately control the concentration of dopants in each stage of an ion-separating apparatus, such that the mobility variations can be controllable and predictable.
Also, a further objective of the present invention is to operate a sequence of ion filters, in which at least one of them is an ion mobility filter, and in which at least one of said ion filters can be operated in transparent mode (allowing the passage of all ions) so as to reduce the time required to identify the peaks of the species of interest. This approach is new and it is part of the present invention.
Yet another objective of the present invention is to identify the filtering parameters of a sequence of ion filters, in which at least one ion filter is an IMS, and in which at least one ion mobility filter can be operated in transparent mode. Another objective of the present invention is to identify the filtering parameters of a sequence of ion filters, in which at least one ion filter is an IMS, and in which at least one ion mobility filter of the type that produces a continuous output of selected ions, and which can also operate in transparent mode, including the VEFMA.